Project Summary RNA-guided, DNA-targeting endonucleases derived from bacterial CRISPR systems promise to rapidly advance genome editing-based therapeutics. This approach, however, is significantly limited by the paucity of available programmable endonucleases. A protein known as CasX was recently discovered by the Doudna group at UC Berkeley and shown to function in a fashion conceptually similar to the unrelated CRISPR proteins Cas9 and Cpf1. The genome editing activity of CasX has not been tested in human cells. Here, in Phase I feasibility studies, we propose to verify and quantify the activity of CasX for genome editing applications. Initial work and analysis of CasX suggests that its activity may be lower than that of Cas9 or Cpf1. To this end, we have developed a high-throughput screening platform for identifying proteins with higher activity. This platform will be used to engineer more active variants of CasX and the most promising variants will be screened for improved activity in human cells. If successful, these experiments will advance a core technology for genome editing that will be rigorously characterized and optimized in an application-oriented Phase II.